U.S. patent number 9,040,242 [Application Number 13/553,256] was granted by the patent office on 2015-05-26 for method to amplify nucleic acids to generate fluorescence labeled fragments of conserved and arbitrary products.
This patent grant is currently assigned to E I DUPONT DE NEMOURS AND COMPANY. The grantee listed for this patent is Anjana Agarwal, Mark A. Jensen. Invention is credited to Anjana Agarwal, Mark A. Jensen.
United States Patent |
9,040,242 |
Agarwal , et al. |
May 26, 2015 |
Method to amplify nucleic acids to generate fluorescence labeled
fragments of conserved and arbitrary products
Abstract
Disclosed herein are methods for the identification of the
species, serotype, and strain of a microorganism. Also disclosed
are primers for use in detecting such microorganisms and kits
comprising such primers.
Inventors: |
Agarwal; Anjana (Wilmington,
DE), Jensen; Mark A. (West Chester, PA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Agarwal; Anjana
Jensen; Mark A. |
Wilmington
West Chester |
DE
PA |
US
US |
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Assignee: |
E I DUPONT DE NEMOURS AND
COMPANY (DE)
|
Family
ID: |
46584402 |
Appl.
No.: |
13/553,256 |
Filed: |
July 19, 2012 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130029341 A1 |
Jan 31, 2013 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61511367 |
Jul 25, 2011 |
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Current U.S.
Class: |
435/6.12;
435/6.11; 435/6.1 |
Current CPC
Class: |
C12Q
1/6846 (20130101); C12Q 1/689 (20130101); C12Q
1/6846 (20130101); C12Q 2527/101 (20130101); C12Q
2535/139 (20130101); C12Q 1/686 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12P 19/34 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Zootecnia et al., "Random amplified polymorphic DNA (RAPD)
interpretation requries a sensitive method for the detection of
amplified DNA," Electrophoresis, 1996, vol. 17, pp. 1553-1554.
cited by examiner .
Grundmann et al., "Automated laser fluorescence analysis of
randomly amplified polymorphic DNA: a rapid method for
investigating nosocomial transmission of Acinetobacter baumannii,"
J. Med. Microbiol., 1995, vol. 43, pp. 446-451. cited by examiner
.
Jorgenson et al., Capillary Electrophoresis: An Introduction,
Methods 4:179-90 (1992). cited by applicant .
Williams et al., DNA polymorphisms amplified by arbitrary primers
are useful as genetic markers, Nucleic Acids Res. 18:6531-34
(1990). cited by applicant .
Chang et al: "FluoMEP: A new genotyping method combining the
advantages of randomly amplified polymorphic DNA and amplified
fragment length polymorphism", Electrophoresis, vol. 28, No. 4, pp.
525-534, Feb. 1, 2007. cited by applicant .
Cladera, et al., "Comparative Genetic Diversity of Pseudomonas
stutzeri Genomovars, Clonal Structure, and Phylogeny of the
Species", Journal Of Bacteriology, vol. 186, No. 16, pp. 5239-5248,
Aug. 3, 2004. cited by applicant .
El Aila, et al., Identification and genotyping of bacteria from
paired vaginal and rectal samples from pregnant women indicates
similarity between vaginal and rectal microflore, BMC Infectious
Diseases, Biomed Central, vol. 9, No. 1, pp. 167-179, Oct. 14,
2009. cited by applicant .
Jensen et al., "Use of Homoduplex Ribosomal DNA Spacer
Amplification Products and Heteroduplex Cross-Hybridization
Products in the Identification of Salmonella Serovars", Applied and
Environmental Microbiology, pp. 2741-2746 , Aug. 1, 1996. cited by
applicant .
Li et al., "Bacterial strain typing in the genomic era", FEMS
Microbiology Reviews, vol. 33, No. 5, pp. 892-916, Sep. 1, 2009.
cited by applicant .
Postlethwait et al., "Using random amplified polymorphic DNAs in
zebrafish genomic analysis.", Methods In Cell Biology, vol. 60, pp.
165-179, 1999. cited by applicant .
Valentini et al., "Random amplified polymorphic DNA (RAPD)
interpretation requires a sensitive method for the detection of
amplified DNA", Electrophoresis, vol. 17, No. 10, pp. 1553-1554,
Jan. 1, 1996. cited by applicant .
Corresponding International Search Report and Written Opinion in
PCT/US2012/047376, mailed Oct. 8, 2012. cited by applicant.
|
Primary Examiner: Kim; Young J
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. Provisional Application
Ser. No. 61/511,367, filed Jul. 25, 2011, which is incorporated
herein by reference in its entirety.
Claims
What is claimed is:
1. A method for the identification of the species, serotype, and
strain of a microorganism comprising: (a) amplifying DNA comprising
variable sequences interspersed between highly conserved rDNA
sequences by PCR and amplifying additional genomic sequences by
random amplified polymorphic DNA (RAPD) PCR using a first primer of
13-14 bases in length and a second primer of 11-13 bases in length,
said first primer comprising: (i) 11-12 contiguous bases from a
highly conserved 16S rDNA region; (ii) 2 bases at the 5' end of
said first primer that are not complementary to said highly
conserved 16S rDNA region; and (iii) a fluorescent label; and (b)
separating the amplified DNA produced in step (a).
2. The method of claim 1, wherein said first primer is a forward
primer.
3. The method of claim 2, wherein said first primer comprises SEQ
ID NO:1.
4. The method of claim 3, wherein said first primer is SEQ ID
NO:2.
5. The method of claim 1, wherein said second primer comprises
11-13 contiguous bases from 23S rDNA.
6. The method of claim 5, wherein said second primer is SEQ ID NO:3
or SEQ ID NO:4.
7. The method of claim 1, wherein said amplifying step utilizes a
third primer of 11-13 bases in length, said third primer comprising
11-13 contiguous bases from 23S rDNA, wherein said third primer is
a different length than said second primer, and wherein one of
either the second primer or third primer comprises 13 contiguous
bases from 23S rDNA.
8. The method of claim 1, wherein step (b) is accomplished by
capillary electrophoresis.
Description
FIELD OF INVENTION
The field relates to methods of microorganism identification using
random amplified polymorphic DNA (RAPD) PCR.
BACKGROUND OF INVENTION
The experimental approach of using short, conserved ribosomal
primers to generate both conserved rDNA fragments and arbitrary
amplification products is presented in U.S. Pat. No. 5,753,467.
Microbial identification at the level of genus and species is
accomplished by the characterization of variations in length and
number of fragments located between highly conserved rDNA
sequences. The level of identification is extended to the level of
serotype and strain by the concurrent amplification of additional
arbitrary regions of the microbial genome. These arbitrary
amplification events are referred to as Random Amplified
Polymorphic DNA (RAPD).
The advantage of this approach is that the same group of primers
are used for all species of microorganism to generate amplification
products. Since the sequences of these primers are highly conserved
among prokaryotic organisms, these primers are generically applied.
A substantial savings of time and expense is realized because the
necessity for screening or presumptive identification has been
eliminated.
The rDNA genetic locus is a genetic unit, which is found in
prokaryotic cells. The conserved amplification targets are those
sequences found in the spacer region between the 16S and 23S
regions of the rDNA genetic locus. These targets are amplified from
conserved sequences in the adjacent 16S and 23S regions.
Significant portions of the nucleic acid sequence, which make up
this genetic locus, are common to all prokaryotic organisms (FIG. 1
shows a generalized schematic of this locus). The overall
relatedness of the 16S, 23S, and 5S regions of this genetic locus
has been used as a tool to classify differing species of
prokaryotes.
The approach described in U.S. Pat. No. 5,753,467 makes use of
short primers of 10-12 bases in length. The products generated by
these primers are separated through the use of an electrophoretic
separation in either agarose or polyacrylamide. The fragments are
then visualized through staining with ethidium bromide. During the
gel loading process, the PCR products could potentially contaminate
the laboratory environment.
SUMMARY OF INVENTION
One aspect is for a method for the identification of the species,
serotype, and strain of a microorganism comprising: (a) amplifying
DNA comprising variable sequences interspersed between highly
conserved rDNA sequences by PCR and amplifying additional genomic
sequences by random amplified polymorphic DNA (RAPD) PCR using a
first primer of 13-15 bases in length and a second primer of 11-13
bases in length, said first primer comprising: (i) at least 11
contiguous bases from a highly conserved 16S rDNA region; and (ii)
a fluorescent label; and (b) separating the amplified DNA produced
in step (a).
Another aspect is for an isolated polynucleotide selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, and the full-length complements thereof.
A further aspect is for a kit comprising a set of primers
comprising PCR primers SEQ ID NO:2 labeled with a fluorophore and
at least one of SEQ ID NO:3 and SEQ ID NO:4.
Other objects and advantages will become apparent to those skilled
in the art upon reference to the detailed description that
hereinafter follows.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic of an rDNA operon disclosed herein.
FIG. 2 shows electropherograms of PCR products generated with 6-FAM
labeled 17mers alone and the with 6-FAM labeled 13 and 11 mer
primer group for Salmonella infantis, Staphylococcus epidermidis,
and Enterobacter aerogenes.
BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES
The following sequences comply with 37 C.F.R.
.sctn..sctn.1.821-1.825 ("Requirements for Patent Applications
Containing Nucleotide Sequences and/or Amino Acid Sequence
Disclosures--the Sequence Rules") and are consistent with World
Intellectual Property Organization (WIPO) Standard ST.25 (1998) and
the sequence listing requirements of the European Patent Convention
(EPC) and the Patent Cooperation Treaty (PCT) Rules 5.2 and
49.5(a-bis), and Section 208 and Annex C of the Administrative
Instructions. The symbols and format used for nucleotide and amino
acid sequence data comply with the rules set forth in 37 C.F.R.
.sctn.1.822.
SEQ ID NO:1 is a forward primer containing an 11 bp sequence from
16S rDNA.
SEQ ID NO:2 is a forward primer containing SEQ ID NO:1 plus two
extra nucleotides at the 5' end.
SEQ ID NO:3 is a reverse primer containing 11 bp from 23S rDNA.
SEQ ID NO:4 is a reverse primer containing 13 bp from 23S rDNA.
SEQ ID NO:5 is a forward primer containing 17 bp from 16S rDNA.
SED ID NO:6 is a reverse primer containing 17 bp from 23S rDNA.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Applicants specifically incorporate the entire contents of all
cited references in this disclosure. Further, when an amount,
concentration, or other value or parameter is given as either a
range, preferred range, or a list of upper preferable values and
lower preferable values, this is to be understood as specifically
disclosing all ranges formed from any pair of any upper range limit
or preferred value and any lower range limit or preferred value,
regardless of whether ranges are separately disclosed. Where a
range of numerical values is recited herein, unless otherwise
stated, the range is intended to include the endpoints thereof, and
all integers and fractions within the range. It is not intended
that the scope of the invention be limited to the specific values
recited when defining a range.
In this disclosure, a number of terms and abbreviations are used.
The following definitions apply unless specifically stated
otherwise.
As used herein, the articles "a", "an", and "the" preceding an
element or component of the invention are intended to be
nonrestrictive regarding the number of instances (i.e.,
occurrences) of the element or component. Therefore "a", "an" and
"the" should be read to include one or at least one, and the
singular word form of the element or component also includes the
plural unless the number is obviously meant to be singular.
The term "comprising" means the presence of the stated features,
integers, steps, or components as referred to in the claims, but
does not preclude the presence or addition of one or more other
features, integers, steps, components or groups thereof. The term
"comprising" is intended to include embodiments encompassed by the
terms "consisting essentially of" and "consisting of". Similarly,
the term "consisting essentially of" is intended to include
embodiments encompassed by the term "consisting of".
As used herein, the term "about" modifying the quantity of an
ingredient or reactant employed refers to variation in the
numerical quantity that can occur, for example, through typical
measuring and liquid handling procedures used for making
concentrates or use solutions in the real world; through
inadvertent error in these procedures; through differences in the
manufacture, source, or purity of the ingredients employed to make
the compositions or carry out the methods; and the like. The term
"about" also encompasses amounts that differ due to different
equilibrium conditions for a composition resulting from a
particular initial mixture. Whether or not modified by the term
"about", the claims include equivalents to the quantities.
Where present, all ranges are inclusive and combinable. For
example, when a range of "1 to 5" is recited, the recited range
should be construed as including ranges "1 to 4", "1 to 3", "1-2",
"1-2 & 4-5", "1-3 & 5", and the like.
The terms "amplification" or "amplify" as used herein include
methods for copying a target nucleic acid, thereby increasing the
number of copies of a selected nucleic acid sequence. Amplification
may be exponential or linear. A target nucleic acid may be either
DNA or RNA. The sequences amplified in this manner form an
"amplicon".
The term "nucleic acid" refers to a polymer of ribonucleic acids or
deoxyribonucleic acids, including RNA, mRNA, rRNA, tRNA, small
nuclear RNAs, cDNA, DNA, PNA, RNA/DNA copolymers, or analogues
thereof. Nucleic acids may be obtained from a cellular extract,
genomic (gDNA) or extragenomic DNA, viral RNA or DNA, or
artificially/chemically synthesized molecules.
The term "complementary" refers to nucleic acid sequences capable
of base-pairing according to the standard Watson-Crick
complementary rules, or being capable of hybridizing to a
particular nucleic acid segment under relatively stringent
conditions. Nucleic acid polymers are optionally complementary
across only portions of their entire sequences.
The term "target", "target sequence", or "target nucleotide
sequence" refers to a specific nucleic acid sequence, the presence,
absence or abundance of which is to be determined.
As used herein, a "primer" for amplification is an oligonucleotide
that is complementary to a target nucleotide sequence and leads to
addition of nucleotides to the 3' end of the primer in the presence
of a DNA or RNA polymerase. The 3' nucleotide of the primer should
generally be identical to the target sequence at a corresponding
nucleotide position for optimal expression and amplification. The
term "primer" as used herein includes all forms of primers that may
be synthesized including peptide nucleic acid primers, locked
nucleic acid primers, phosphorothioate modified primers, labeled
primers, and the like. As used herein, a "forward primer" is a
primer that is complementary to the anti-sense strand of dsDNA. A
"reverse primer" is complementary to the sense-strand of dsDNA.
Primers are typically between about 10 and about 100 nucleotides in
length, preferably between about 15 and about 60 nucleotides in
length, and most preferably between about 20 and about 30
nucleotides in length.
An oligonucleotide (e.g., a probe or a primer) that is specific for
a target nucleic acid will "hybridize" to the target nucleic acid
under suitable conditions. As used herein, "hybridization" or
"hybridizing" refers to the process by which an oligonucleotide
single strand anneals with a complementary strand through base
pairing under defined hybridization conditions. "Specific
hybridization" is an indication that two nucleic acid sequences
share a high degree of complementarity. Specific hybridization
complexes form under permissive annealing conditions and remain
hybridized after any subsequent washing steps. Permissive
conditions for annealing of nucleic acid sequences are routinely
determinable by one of ordinary skill in the art and may occur, for
example, at 65.degree. C. in the presence of about 6.times.SSC.
Stringency of hybridization may be expressed, in part, with
reference to the temperature under which the wash steps are carried
out. Such temperatures are typically selected to be about 5.degree.
C. to about 20.degree. C. lower than the thermal melting point (Tm)
for the specific sequence at a defined ionic strength and pH. One
set of preferred conditions uses a series of washes starting with
6.times.SSC, 0.5% SDS at room temperature for 15 min, then repeated
with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30 min, and then
repeated twice with 0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30
min. A more preferred set of conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times.SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of
stringent hybridization conditions is 0.1.times.SSC, 0.1% SDS,
65.degree. C. and washed with 2.times.SSC, 0.1% SDS followed by a
final wash of 0.1.times.SSC, 0.1% SDS, 65.degree. C.
The term "label" refers to any detectable moiety. A label may be
used to distinguish a particular nucleic acid from others that are
unlabeled, or labeled differently, or the label may be used to
enhance detection.
The term "specimen" means a biological sample such as saliva,
stools, urine, blood, gastric biopsy, gastrointestinal tissue,
tumor cells, mucus secretions, dental plaque, and other biological
tissues; meat products; food products; and environmental samples
such as soil or water.
Nucleic Acid Detection
To avoid the potential hazard of laboratory environment
contamination, the present method requires the use of fluorescence
labeled primers. Products generated from these primers can be
directly detected by capillary electrophoresis. Use of short
fluorescence labeled primers, 10 to 12 bases, presents a difficulty
because the presence of the fluorescent moiety makes such primers a
poor substrate for DNA polymerases. Longer primers with 100%
homology to the conserved sequences cannot be substituted because
such primers will amplify only the ribosomal fragments without the
arbitrarily primed pattern elements that are critical to strain
level differentiation.
The minimum length required for incorporation of a fluorescence
labeled primer was 13 bases. Since 13-base primers with a perfect
match to the ribosomal site amplified only the ribosomal fragments,
it was necessary to employ a 2-base mismatch on the 5' end of the
fluorescence labeled primer. Since only the last 11 bases matched
the ribosomal sequence, such primers are capable of amplifying both
ribosomal fragments and arbitrary genomic fragments
simultaneously.
More particularly, the present method comprises amplifying DNA
comprising variable sequences interspersed between highly conserved
rDNA sequences by PCR and amplifying additional genomic sequences
by RAPD PCR using a first primer of 13-15 bases in length and a
second primer of 11-13 bases in length. The first primer comprises
at least 11 contiguous bases from a highly conserved 16S rDNA
region and a fluorescent label. In a second step, the method
comprises separating the amplified DNA produced in the amplifying
step.
The method described herein is useful in identifying a wide variety
of microorganisms. Representative but not exhaustive of the many
types of organisms including both genus, species and serotype that
may be elicited through the use of the present procedures are
Listeria monocytogenes, Listeria welshimeri, Listeria innocua,
Listeria ivanovii, Salmonella typhimurium, Salmonella enteritidis,
Salmonella newport, Salmonella infantis, Staphylococcus aureus,
Staphylococcus scuiri, Staphylococcus warneri, Staphylococcus
saprophyticus, Staphylococcus epidermidus, Escherichia coli,
Escherichia fergusonii, Escherichia blattae, Escherichia hermanii,
Escherichia vulneris, Citrobacter freundii, Citrobacter diversus,
Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter
cloacae, Proteus mirabilis, Proteus vulgaris, and Yersinia
enterocolitica. Such a listing may form a database of previously
visualized products which when compared to the electrophoresed,
visualized fragment products according to the present method,
afford an identification of the species (and the serotype and
strain if applicable).
It is readily appreciated by one skilled in the art that the
present method may be applied to microorganisms in the context of a
wide variety of circumstances. Thus, a preferred use of the present
invention is in the identification of microorganisms in food.
Additionally, research directed to microbial infections in humans,
other animals, and plants would benefit from the procedure
herein.
Nucleic acids may be isolated from a sample according to any
methods well known to those of skill in the art. If necessary the
sample may be collected or concentrated by centrifugation and the
like. The cells of the sample may be subjected to lysis, such as by
treatments with enzymes, heat, surfactants, ultrasonication, or
combination thereof.
Various methods of nucleic acid extraction are suitable for
isolating nucleic acids. Suitable methods include phenol and
chloroform extraction. See, e.g., Maniatis et al., Molecular
Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory
Press (1989).
RAPD PCR is disclosed in U.S. Pat. No. 5,126,239 (see also,
Williams et al., Nucleic Acids Res. 18:6531-34 (1990)). The
approach describes the use of a small oligonucleotide, i.e.,
greater than seven nucleotides, of arbitrary composition in a DNA
amplification reaction. Short primers are used in order that
complementary and reverse complementary sequences to the primer can
be found at distances along the genome which are sufficiently small
that DNA amplification can take place. The fragments generated in
the amplification process are called RAPD markers. These RAPD
markers show a size distribution which is sensitive to modest
differences in the genomic makeup of the DNA used in the
amplification process.
As noted in U.S. Pat. No. 5,753,467, the process of U.S. Pat. No.
5,126,239 requires 45 cycles, which frequently results in the
formation of secondary amplification products and nonspecific DNA
synthesis. A product profile background which contains high levels
of such secondary amplification products and nonspecific DNA can
severely restrict the ability of pattern recognition software to
compare such a product profile with a known database. The process
disclosed herein, however, uses fewer amplification cycles with
longer annealing times to produce a far less complex product
profile with a significantly reduced nonspecific DNA
background.
The skilled artisan is capable of designing and preparing arbitrary
primers that are appropriate for RAPD PCR. The length of the
amplification primers depends on several factors including the
nucleotide sequence identity and the temperature at which these
nucleic acids are hybridized or used during in vitro nucleic acid
amplification. The considerations necessary to determine a
preferred length for an amplification primer of a particular
sequence identity are well known to the person of ordinary skill in
the art.
Primers that amplify a nucleic acid molecule can be designed using,
for example, a computer program such as OLIGO (Molecular Biology
Insights, Inc., Cascade, Colo.). Important features when designing
oligonucleotides to be used as amplification primers include, but
are not limited to, an appropriate size amplification product to
facilitate detection (e.g., by electrophoresis), similar melting
temperatures for the members of a pair of primers, and the length
of each primer (i.e., the primers need to be long enough to anneal
with sequence-specificity and to initiate synthesis but not so long
that fidelity is reduced during oligonucleotide synthesis).
Preferred primers, along with their targets, are described in Table
1 below.
As discussed in U.S. Pat. No. 5,753,467, a significant degree of
intramolecular hybridization is known to occur within the rDNA
genetic locus. The resulting secondary structure frequently makes
it difficult for amplification primers to compete for hybridization
sites. In order to enhance the amplification of fragments contained
within the rDNA region it is necessary to modify the amplification
temperature profile which is typically practiced. The principal
modifications consist of the use of substantially longer annealing
times, in a range of about 3 to about 7 minutes. Amplification
reactions are being run under high stringency conditions in
conjunction with a decreased number of amplification cycles. A high
stringency amplification is accomplished by running the reaction at
the highest annealing temperature where products are reproducibly
formed. Use of maximum annealing temperature insures that only the
most stable hybridization structures will form and that the areas
surrounding the priming sites will possess a minimal amount of
secondary structure.
The presence or absence target nucleic acids can determined, e.g.,
by analyzing the amplified nucleic acid products of the primer
extension by size using standard methods, for example, agarose gel
electrophoresis, polyacrylamide gel electrophoresis, capillary
electrophoresis, pulsed field electrophoresis, denatured gradient
gel electrophoresis, DNA microarrays, or mass spectrometry.
Preferably, capillary electrophoresis is used to separate the
amplified products.
In capillary electrophoresis, the length of a nucleic acid fragment
is examined by allowing a sample to migrate through a thin tube
filled with gel and measuring a period of time required for the
sample to migrate a certain distance (e.g., to the end of a
capillary). Upon capillary electrophoresis, it is usual to detect a
sample using a fluorescence signal detector that is installed at
the end of a capillary.
Apparatuses for carrying out capillary electrophoresis are
well-known. Many references are available describing the basic
apparatus and several capillary electrophoresis instruments are
commercially available, e.g., from Applied Biosystems (Foster City,
Calif.). Exemplary references describing capillary electrophoresis
apparatus and their operation include Jorgenson, Methods 4:179-90
(1992); Colburn et al., Applied Biosystems Research News, issue 1
(winter 1990); and the like.
With respect to fluorescence measurement, when PCR is performed
using primers labeled at their 5' ends with a fluorophore, the
amplified target sequence is labeled with the detectable
fluorescent material, and the intensity of fluorescence emitted
from the fluorescent material is measured using a fluorescence
spectrophotometer. Suitable fluorophores include, but are not
limited to, 6-FAM; Alexa fluor 405, 430, 488, 532, 546, 555, 568,
594, 633, 647, or 660; Cy2; Cy3; Cy3.5; Cy5; Cy5.5; Cy7;
hydroxycoumarin; methoxycoumarin; aminocoumarin; fluorescein; HEX;
R-phycoerythrin; rhodamine Red-X; ROX; Red 613; Texas Red;
allophycocyanin; TruRed; BODIPY 630/650; BODIPY 650/665; BODIPY-FL;
BODIPY-R6G; BODIPY-TMR; BODIPY-TRX; carboxyfluorescein; Cascade
Blue; 6-JOE; Lissamine rhodamine B; Oregon Green 488, 500, or 514;
Pacific Blue; REG; Rhodamine Green; SpectrumAqua; TAMRA; TET; and
Tetramethylrhodamine.
As discussed above, preferred primers are disclosed in Table 1. One
embodiment related thereto is for an isolated polynucleotide
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, and SEQ ID NO:4. Another embodiment is for kit comprising
a set of primers comprising PCR primers SEQ ID NO:2 labeled with a
fluorophore and at least one of SEQ ID NO:3 and SEQ ID NO:4. In
some aspects, the kit comprises both PCR primers SEQ ID NOs: 3 and
4.
Such a kit may comprise a carrier being compartmentalized to
receive in close confinement therein one or more container means,
such as tubes or vials. One of said container means may contain
unlabeled or detectably-labeled primers. The primers may be present
in lyophilized form or in an appropriate buffer as necessary. One
or more container means may contain one or more enzymes or reagents
to be utilized in PCR reactions. These enzymes may be present by
themselves or in admixtures, in lyophilized form or in appropriate
buffers. The kit may also contain some or all the additional
elements necessary to carry out the PCR and/or CE, such as buffers,
extraction reagents, enzymes, pipettes, plates, nucleic acids,
nucleoside triphosphates, filter paper, gel materials, transfer
materials, autoradiography supplies, and the like.
GENERAL METHODS
The following examples are provided to demonstrate preferred
embodiments. It should be appreciated by those of skill in the art
that the techniques disclosed in the examples which follow
represent techniques discovered by the inventor to function well in
the practice of the methods disclosed herein, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
presently disclosed methods.
The following abbreviations in the specification correspond to
units of measure, techniques, properties, or compounds as follows:
"sec" or "s" means second(s), "min" means minute(s), "h" or "hr"
means hour(s), ".mu.L" means microliter(s), "mL" means
milliliter(s), "L" means liter(s), ".mu.M" means micromolar, "M"
means molar, "pmol" means picomole(s), "g" means gram(s), ".mu.g"
means microgram(s), "ng" means nanogram(s), "pg" means picogram(s),
"CE" means capillary electrophoresis, "bp" means basepair(s),
"6-FAM" means 6-carboxyfluorescein.
EXAMPLE 1
Initially, amplification reactions were carried out with a 17-mer
primer pair (SEQ ID NOs: 5 and 6), one of which contained a 6-FAM
fluorescent tag (SEQ ID NO:5), to determine the size distribution
of the ribosomal component of the PCR amplification. These
fragments provided reference products that made it possible to
identify the ribosomal pattern components in the mixed ribosomal
and arbitrary amplification.
To generate the ribosomal/RAPD DNA profile, a mixture of three
individual primers (one forward and two reverse primers) were used.
The forward primer was labeled with 6-FAM dye and contained an 11
bp sequence from 16S rDNA (SEQ ID NO:1) plus two extra nucleotides
(CA) added at the 5' end (SEQ ID NO:2). The CA sequence does not
match the known conserved 16S ribosomal sequence and serves only to
make the fluorescence labeled primer a better substrate for the DNA
polymerase. To amplify both ribosomal and RAPD fragments, sequences
of both reverse primers were obtained from a single location of 23S
rDNA. The sequences of the reverse primers are as follows: R-23S
reverse primer 1: SEQ ID NO:3 (11 mer) and R-23S reverse primer 2:
SEQ ID NO:4 (13 mer).
TABLE-US-00001 TABLE 1 Primers Location of primers Sequence 5'-3'
16S-17mer 6-FAM label (6-FAM)-SEQ ID NO: 5 23S-17mer SEQ ID NO: 6
23S-11mer SEQ ID NO: 3 23S-13mer SEQ ID NO: 4 16S-13mer 6-FAM
(6-FAM)-SEQ ID NO: 2
The PCR reaction was performed in 30 .mu.l of reaction mixture,
contained 1.times.PCR buffer from KAPA 2G robust HotStart ReadyMix
(Kapa Biosystems, Woburn, Mass.), 20 pmol of forward primer
(6-FAM)-SEQ ID NO:2), 20 pmol of reverse primer1 (SEQ ID NO:3), 4
pmol of reverse primer 2 (SEQ ID NO:4), and 50 pg to 100 ng of
gDNA. The PCR fragments were amplified by initial denaturation at
95.degree. C. for 2 min., followed by 35 cycles of (95.degree.
C.-30 sec., 45.degree. C.-5 min., and 72.degree. C.-30 sec.). The
final extension was performed at 72.degree. C. for 10 min. GeneScan
1200 LIZ.RTM. (Applied Biosystems, Carlsbad, Calif.) was used as a
DNA size standard and was added in the PCR product after
amplification. The PCR fragments were separated and detected using
an Applied Biosystem's model 3730 CE instrument. Applied Biosystem
PeakScanner software was used to identify fragment peaks in the CE
pattern and characterize them.
Examples of three amplification reactions are shown in FIG. 2.
Reactions were carried out as described in the above PCR protocol
for genomic DNA extracts from Salmonella infantis, Staphylococcus
epidermidis, and Enterobacter aerogenes. In FIG. 2, each
electropherogram is compared to an electropherogram that contains
only the ribosomal spacer fragments. This is done to demonstrate
that these ribosomal fragments are preserved in the presence of the
arbitrary amplification events and to show that yields of arbitrary
and ribosomal fragments were comparable.
The approach of combining 11-base and 13-base primers, one of which
contains a fluorescent tag, clearly provides amplification of both
ribosomal and RAPD fragments. Under the specified amplification
conditions, yields of the ribosomal and dominant RAPD fragments are
comparable.
The resulting patterns contain fluorescence labeled fragments that
can be separated by capillary electrophoresis to produce a pattern
of products that contain both conserved ribosomal fragments and
arbitrary RAPD fragments.
SEQUENCE LISTINGS
1
6111DNAArtificial SequencePrimer 1gaagtcgtaa c 11213DNAArtificial
SequencePrimer 2cagaagtcgt aac 13311DNAArtificial SequencePrimer
3aaggcatcca c 11413DNAArtificial SequencePrimer 4caaggcatcc acc
13517DNAArtificial SequencePrimer 5gtgaagtcgt aacaagg
17617DNAArtificial SequencePrimer 6caaggcatcc accgtgt 17
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